Protease Antigen Retrieval Protocol

 

 

 

Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The protease based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

 

Solutions and Reagents:  

     

Protease Solution (0.05% in Distilled Water)

Protease ----------------------------------- 5 mg

      Distilled water --------------------------- 10 ml

      Mix to dissolve. Adjust to pH 7.8 using 1N NaOH. Store at -20 ºC.

 

Procedure:

  1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.

  2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.

  3. Rinse in distilled water.

  4. Cover sections with protease working solution and incubate for 10-20 minutes at 37 °C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user).

  5. Allow sections to cool at room temperature for 10 minutes.
  6. Rinse sections in PBS Tween 20 for 2x2 min.
  7. Block sections with for 30 minutes.
  8. Perform avidin/biotin blocking if necessary.
  9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C.
  10. Rinse sections with PBS Tween 20 for 2x2 min.
  11. Block sections with peroxidase blocking solution for 10 minutes.
  12. Rinse with PBS Tween 20 for 3x2 min.
  13. Proceed to standard immunohistochemistry protocol.

Notes: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application.

 

References:

 

Battifora H, Kopinski M (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J Histochem Cytochem. 34(8):1095-100. PubMed Abstract